Inducing cytotoxic T lymphocyte responses

ABSTRACT

The invention provides compositions and methods for inducing MHC class I-restricted cytotoxic T lymphocyte responses in a mammalian host by immunization with non-replicating protein antigens. The compositions of the invention comprise a particulate-protein complex capable of inducing a class I-restricted CTL response to a protein antigen in a mammal, in which the particulate protein complex comprises a particulate component having an average diameter ranging in size from about 0.5 μm to about 6 μm, linked to a non-replicating protein antigen derived from a tumor cell or from pathogenic organism where CTL response is likely to play an important role in conferring protective immunity in a mammal, with the proviso that the particulate component is not a prokaryotic or eukaryotic cell or a micellar multimicellar, or liposome vesicle composed of detergents or lipids. The non-replicating protein antigen is attached to the particle component through a covalent or non-covalent association to form particulate protein antigen complexes and the complexes are administered to a mammalian host in conjunction with a pharmaceutically acceptable excipient, in a CTL-stimulatory amount. The invention also provides non-replicating vaccines and methods of vaccinating a mammalian host against pathogenic diseases or tumors for CTL immunity.

STATEMENT OF GOVERNMENT RIGHTS TO INVENTION

This invention was made with United States government support underGrant R01 AI 31337, awarded by the National Institutes of Health. TheUnited States government therefore has certain rights to the invention.

STATEMENT OF RELATED APPLICATION

This application is a continuation-in-part of application U.S. Ser. No.08/003,233, filed on Jan. 11, 1993, abandoned.

FIELD OF THE INVENTION

This invention relates to compositions and methods for inducing class Imajor histocompatibility-restricted cytotoxic T lymphocyte responses ina mammal by immunization with particulate protein antigen complexescomprising non-replicating protein antigen. The invention also relatesto vaccine development for cytotoxic T lymphocyte immunity and methodsof treating infectious diseases.

BACKGROUND OF THE INVENTION

One of the major immune responses that protects a host from disease,especially from intracellular infection, is the generation of cytotoxicT lymphocytes (CTLs). CTLs kill host cells that are infected and therebyeliminate the production and/or reservoir of the pathogen. CTLs may alsocontrol secondary pathogenic effects induced by infectious organisms,for example, the growth of transformed cells. There is abundant evidencethat CTLs are critical components in the defense of the host againstseveral viral pathogens, including influenza, POX and Herpes (Blanden,Transplant Rev., 19:56 (1975); Yap et al., Nature, 273:238 (1978).Furthermore, CTLs can provide immunity in vivo to retrovirally-induceddiseases (Earl et al., Science, 234:728 (1986)). There is increasingevidence that CTLs may play a role in protection from humanimmunodeficiency virus (HIV). For example, CTLs from infected humans andapes can lyse infected cells and inhibit virus production in vitro.

One of the most efficacious and cost effective therapies for theprevention of infectious diseases is the stimulation of specific immuneresponse through vaccination. The need for an effective HIV vaccine istragically apparent.

Historically, vaccines have been prepared by killing or attenuating apathogen, such as a virus or bacterium, and then injecting the resultingparticles into a patient or host animal. Vaccines have also beenprepared by using only a portion of the pathogenic organism, such as apredominantly important protein subunit from a bacterium or virus. Whilenon-living virus particles and subunit vaccines can prime for a class-IIrestricted antibody response, such non-living vaccines typically do notprime for cytotoxic T lymphocyte immunity.

It is now well established that the tight segregation of the MHC class Ipathway of antigen presentation accounts for the failure of mostprotein-based vaccines to prime CTL responses and is a major obstacle tousing such vaccines. Specifically, from the published literature it isknown that most antigens in the extracellular fluid are taken up byspecialized antigen presenting cells (APCs), processed in an endosomalcompartment, and subsequently displayed in association with class II MHCmolecules, which elicits an antibody response. In contrast, mostexogenous antigens are not presented in association with class I MHCmolecules, which is necessary for a class I-restricted CTL response.However, exogenous antigens are presented in association with class Imolecules if they are introduced, via experimental manipulations, intothe cytoplasm ("cytosol") of cells. It is thought that the antigenicpeptides that arise from processing in the cytosol are transported tothe endoplasmic reticulum where they associate with class I MHCmolecules. The failure of exogenous proteins to be presented inassociation with class I molecules reflects the inability of theseproteins and their degraded endosomal products, to communicate with theappropriate cytosolic compartment, under physiological conditions. As aconsequence of this segregation between MHC-class I and class IIantigen-presentation pathways, CTLs are selectively targeted topathologically-affected cells (ie. cells synthesizing abnormalproteins). Uninfected, healthy cells are not at risk of elimination whenthey encounter antigens in the extracellular fluids.

Despite the generally recognized inability of most antigens to prime CTLresponse, there have been reports in literature of inducing MHC classI-restricted CTLs with non-replicating antigen in vivo. For example,Zhou et al. have shown that allogeneic splenocytes, MHC-free red bloodcells, and synthetic lipid vesicles (liposomes) loaded with chickenovalbumin (OVA) can elicit an OVA-specific MHC class I-restricted immuneresponse. J. Immunol., 149:1599 (Sep. 1, 1992). Liposomes were also usedby Reddy et. al to incorporate soluble proteins of OVA andβ-galactosidase for priming a CD8⁺ CTL response to antigen in vivo inmice. J. Immunol., 148:1585 (Mar. 1, 1992), while Bevan et al. havedemonstrated CTL priming against soluble OVA with a cell-associatedsystem J. Exp. Med., 171:377 (1990). Complex adjuvants, such as completeFreund's adjuvant (CFA) have also been used with some measure ofsuccess. Bacteria, such as Mycobacterium and Staph aureus have also beenincluded as adjuvants in immunization for CTL response. Randall & Young,J. Virol., 65:719-726 (Feb. 1991).

Immune stimulating complexes (ISCOMS), which are multimicellar complexesof cholesterol, phospholipid, and a saponin, have been used fairlyextensively as carriers of subunit vaccines, especially in veterinaryapplications, and have been shown to induce CD8⁺ MHC class I-restrictedCTL. See, for example, Nadon et al., Vaccine, 10:107 (1992); Mowat etal., J. Immunol. 72:317-322 (1991); Takahashi et al., Nature, 344:873(Apr. 26, 1990); and Morein, Nature, 322:287 (Mar. 17, 1988). Protectiveclass I MHC-restricted CTLs have been also induced in mice by arecombinant influenza vaccine in an aluminum hydroxide adjuvant, whichis currently the only adjuvant licensed by the FDA for clinical use inhumans. Dillon et al., Vaccine, 10:309 (1992).

Although the foregoing preparations have been used to induce classI-restricted CTLs with varying degrees of success, none is withoutpotential disadvantages, and those skilled in the area of vaccinationbiology continue to seek compositions and methods for inducing CTLresponses to non-living proteins to confer protective immunity frompathogenic infection and minimize serious side effects. For example,many of the complex adjuvants that have been shown to induce CTLs inlaboratory animals are unacceptable for use in domestic animals andhumans because of their potential toxicity. Cell-associated formulationsdo not represent a practical immunization strategy for human vaccines,given the significant possibility for infectious contamination in thepreparation. CFA does not routinely achieve priming, even in mice, andmay result in tumor formation and tissue necrosis. Antigen encapsulationin liposomes or in a lipid/detergent-based adjuvant, such as the ISCOMmatrix, appears to be the most promising approach for immunizing withnon-replicating protein, but even this approach may have drawbacks. Forexample, the ISCOM matrix includes a saponin as an essential component.Saponins are hemolysins and there is an indication in the literaturethat at least some saponins may be cytotoxic at immunogenicconcentrations. In addition, the FDA imposes stringent stabilityrequirements on formulations for human vaccines, and cell associatedformulations, liposomes and the ISCOM matrix are potentially problematicfrom that standpoint.

It is an object of the present invention to provide new compositions andmethods for inducing MHC class I-restricted CTLs with non-replicatingantigens.

SUMMARY OF THE INVENTION

This, as well as other objects and advantages, are achieved inaccordance with the present invention, which provides pharmaceuticalcompositions and methods for inducing MHC class I-restricted cytotoxic Tlymphocyte responses in a mammalian host by immunization withnon-replicating protein antigens. The pharmaceutical compositions of theinvention comprise a two-component complex including a particlecomponent, which is not a prokaryotic or eukaryotic cell, or micellar,multimicellar, or liposome vesicle composed of detergents and/or lipids,ranging in size from about 10 nm to about 50 μm, preferably from 0.5 μmto 6 μm, and a non-replicating protein antigen. The non-replicatingprotein antigen is attached to the particle component through a covalentor non-covalent association to form particulate protein antigencomplexes which are formulated with a pharmaceutically acceptableexcipient to form the pharmaceutical compositions of the invention. Inone embodiment of the invention, the non-replicating protein antigencomprises at least one pathogenrelated protein, especially a bacterialor viral protein. In another embodiment, the non-replicating proteinantigen comprises a tumor antigen.

Another embodiment of the invention provides methods for targetingprotein antigen into the MHC class I pathway of antigen presentation ina subpopulation of antigen presenting cells that are capable ofprocessing exogenous antigen from the extracellular environment andpresenting the processed antigen in association with class I MHCmolecules.

In another embodiment of the invention, the particulate proteincomplexes are used in CTL vaccine development, for example in an assayfor identifying potential CTL vaccines from a plurality of candidatecomplexes.

In yet another embodiment, the invention provides non-replicatingvaccines and methods of vaccinating a mammalian host, especially a humanbeing, for CTL immunity.

The invention is useful in vaccination for CTL immunity with proteinantigens and in vaccine development.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A depicts the results of an antigen presentation assay, whichdemonstrates that accessory cell depletion from splenic antigenpresenting cells removes antigen presenting cells that process andpresent antigen with class I molecules. The closed circles represent theantigen-presenting activity of unfractionated splenocytes; the closedtriangles the antigen-presenting activity of G10-passed spleen cells;and the open circles the activity of sIg+ B cells positively selectedfrom G10-spleen by panning on anti-Ig coated plates. Data are expressedare the mean counts per minute (CPM) of³ H!TdR incorporated by HT-2cells.

FIG. 1B depicts the results of an antigen presenting assay demonstratingthat cells in the low buoyant density fraction of fractionatedsplenocytes are enriched for antigen presenting cells that can processand present exogenous antigen, in this case, chicken ovalbumin (OVA)with class I MHC. The closed circles represent antigen presentation byunfractionated splenocytes; the closed triangles represent the activityof splenocytes in the high density fraction; and the open circlesrepresent the activity of splenocytes in the low density fraction. Thedata are expressed as in FIG. 1A.

FIG. 1C depicts the results of an antigen presentation assay whichdemonstrates that resetting cells in the low buoyant density fraction offractionated splenocytes are enriched for antigen presenting cells thatcan process and present exogenous antigen in association with class IMHC molecules activity. The closed circles represent antigenpresentation activity of unfractionated splenocytes; the open trianglesrepresent the activity of the cells recovered in the low densityfraction; the closed triangles represent the antigen presenting activityof non-rosetting cells in the low density fraction; and the open circlesrepresent the resetting cells. Rosetting was obtained by incubatingcells recovered in the low density fraction with sheep red blood cellssensitized with rabbit anti-sheep red blood cell antibodies. Data areexpressed as in FIG. 1A.

FIG. 1D depicts the results of an antigen presentation assaydemonstrating that activated B cells do not present exogenous antigenwith class I MHC molecules. In this experiment, G10-passed splenocyteswere stimulated with lipopolysaccharide (LPS) prior to the HT-2 assay.The closed circles represent the antigen-presenting activity of the LPSand the open circles the activity of thio-glycolate induced peritonealexudate cells (PECs). Data are expressed as the mean counts per minute(CPM) of³ H!TdR incorporated by HT-2 cells.

FIG. 2 depicts the results of an analysis of the adherence properties ofthe antigen presenting cells that present exogenous OVA with MHC classI. The antigen presenting activity of the various fractions, i.e.,unfractionated (open triangles), 2 hr nonadherent cells (solid squares),24 hr adherent (open squares) and 24 nonadherent (open circles) wasassayed with RF33.70 T--T hybridomas with or without OVA (4 mg/ml) incultures prepared and assayed as described, except that theunfractionated low density cells and 2 hr nonadherent cells were addedto microtiter plates and incubated for 24 hr before the addition of theT--T hybrids and antigen.

FIG. 3A depicts the results of an antigen presentation assaydemonstrating that antigen presenting cells normally present in theperitoneal cavity can present exogenous antigen (OVA) with class Imolecules. The solid circles represent the antigen presenting activityof unfractionated spleen cells; the open squares represent the antigenpresenting activity of resident peritoneal exudate cells (PECs); theopen triangles represent the antigen presenting activity of peptoneinduced PECs; and the open circles represent the activity ifthioglycolate-induced PECs.

FIG. 3B depicts the results of an antigen presentation assaydemonstrating that the antigen presenting cells normally present in theperitoneal cavity that can present exogenous antigen with class Imolecules are adherent to plastic. The solid circles represent theantigen presenting activity of unfractionated spleen cells; the opentriangles represent the antigen presenting activity of the non-adherentPECs; and the open circles represent the activity of the adherent PECs.

FIG. 4A depicts the results of an antigen presenting assay demonstratingthat covalent linkage of antigen to an iron oxide particle dramaticallyincreases the antigen presenting efficiency of a macrophage clone, A3.1,that presents soluble antigen in association with class I MHC.

FIG. 4B depicts the results of an antigen presenting assay demonstratingthat linkage of antigen to iron oxide or silica particles dramaticallyincreases the antigen presenting efficiency of a macrophage clone, A3.1,that presents soluble antigen in association with class I MHC.

FIG. 4C depicts the results of an antigen presenting assay demonstratingthat non-covalent linkage of antigen to latex particles dramaticallyincreases the antigen presenting efficiency of a macrophage clone, A3.1,that presents soluble antigen in association with class I MHC.

FIG. 5 depicts the results of an antigen presenting assay demonstratingthat the covalent linkage of antigen (OVA) to an iron oxide particledramatically increases the antigen presenting efficiency ofthioglycolate-induced PECs.

FIG. 6A through FIG. 6D depicts the results of a chromium release assaywhich demonstrates the ability of particulate protein antigen complexesto prime CTL responses in vivo. C57BL/6 mice were injectedsubcutaneously with the indicated antigen preparations (OVA particle=OVAcovalently linked to iron oxide), with the amount of antigen indicatedat the top of the Figures. Seven days later, the animals were sacrificedand splenocytes restimulated in vitro with irradiated EG7 cells. Afterfive days of culture, the cells were tested for their ability to lyse ⁵¹Cr-labeled EL4 cells or EG7 cells. The percent specific release ofchromium was measured for the indicated effector to target ratios (E:T).FIGS. 6A and 6B show that the OVA-particles primed CTLs at 90 μg and 18μg of complex, respectively. FIGS. 6C and 6D show that same amounts ofsoluble OVA were not effective.

FIG. 7A through FIG. 7C depicts the results of a chromium release assaywhich demonstrates the ability of particulate protein antigen complexesto prime CTL responses in vivo. C57BL/6 mice were injectedsubcutaneously with the indicated antigen preparations (β-gal=E. coliβ-galactosidase linked to iron oxide particles), with the amount ofantigen indicated at the top of the Figures. Seven days later, theanimals were sacrificed and splenocytes restimulated in vitro withirradiated P13.4 cells. After five days of culture, the cells weretested for their ability to lyse 51Cr-labeled P815 or P13.4 cells. Thepercent specific release of chromium was measured for the indicatedeffector to target ratios (E:T). FIGS. 7A shows that the β-gal-particlesprimed CTLs at 13 μg of the complex. FIGS. 7B and 7C show that sameamount of soluble β-gal and ten times the amount of β-gal, respectively,were ineffective.

FIG. 8A and FIG. 8B show that the protein hen enzyme lysozyme (HEL), canprime CTL's in mice in vivo, when linked to an iron oxide particle inaccordance with the present invention.

FIG. 9 depicts the results of a tumor growth assay which demonstrates ina model of tumor immunity that immunization with ovalbumin conjugated toiron oxide particles inhibits tumor formation in mice subsequentlychallenged with B16 melanoma cells transfected with a cDNA encodingchicken ovalbumin. In the Figure, the open circles represent the miceimmunized with ovalbumin conjugated to the iron oxide, the closedcircles represent the mice immunized with ovalbumin alone, and the opentriangles represent the mice that were unimmunized.

FIG. 10 depicts the results of a survival assay which demonstrates in amodel of tumor immunity that immunization with ovalbumin conjugated toiron oxide particles significantly increases survival of micesubsequently challenged with EL4 lymphoma cells transfected with a cDNAencoding chicken ovalbumin compared to animals that had not beenimmunized. In the Figure, the open triangles represent the miceimmunized with ovalbumin conjugated to the iron oxide and the closedtriangles represent mice that were not immunized.

FIG. 11 depicts the results of a survival assay which demonstrates thatimmunization with a commercially-available inactivated influenza viruspreparation conjugated to iron oxide particles significantly increasedsurvival of mice subsequently challenged intranasally with theinactivated virus compared to animals that had not been immunized. Inthe Figure, the open circles represent the mice immunized withinactivated influenza virus strain A conjugated to the iron oxide andthe closed circles represent mice that were not immunized.

FIGS. 12A and 12B depict the results of a chromium release assay whichdemonstrates the ability of particulate protein antigen complexes toprime CTL responses in vivo in immunodeficient mice. C57BL/6 or MHCclass II mutant mice were injected subcutaneously with ovalbuminconjugated to iron oxide beads (25 μg) Seven days later, one group ofthe mice was reimmunized with the same immunogen. Fourteen days afterthe initial immunization the animals were sacrificed and splenocytesrestimulated in vitro with irradiated EG7 cells. After five days ofculture, the cells were tested for their ability to lyse ⁵¹ Cr-labeledEL4 cells (BG) or EG7 cells. The percent specific release of chromiumwas measured for the indicated effector to target ratios (E:T). FIG. 12Ashows the results for mice primed only one time; FIG. 12B shows theresults for the animals that were reimmunized on day 7. The results showthat the particulate protein antigen complex could prime a CTL response,even in immunodeficient animals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon our discovery of a normal antigenpresenting cell, which is resident in unstimulated lymphoid tissue ofmice, that can take up antigens, process them, and present them inassociation with class I molecules. See, Rock, Science, 249:918-921(August, 1990). We have now characterized this specialized cell as amacrophage, which expresses both class II molecules and the complementreceptor (FcR). Without wishing to be held to any particular theory ormechanism of the invention, it is presently believed that theparticulate antigen complexes of the invention are more efficientlytargeted to the class I pathway of these antigen presenting cells thansoluble protein antigen, especially in vivo, because they are morereadily phagocytized.

The particulate antigen complexes of the invention contain twocomponents: a particle component having an average diameter ranging insize of from about 10 nm to 50 μm; and a non-replicating proteinantigen. The two components are physically linked through a covalent ora non-covalent association.

The particle component can be formed from almost any material that isnot deleterious to the antigen presenting cells or the mammalian host.However, the particle is not a liposome vesicle or a micellar ormultimicellar matrix formed of detergents and lipids, e.g. an ISCOMmatrix, nor is it a prokaryotic or eukaryotic cell or cell debris. Asused herein, the term liposome means a synthetic vesicle or sac composedof a lipid bilayer.

For example, the particle component of the invention can be formed froma natural or synthetic organic polymeric material or an aqueoussuspension or emulsion thereof. Particles are preferably formed frombiocompatible, natural polymers, such as polysaccharides, includingstarches, cellulose, pectin, seaweed gums (agar), vegetable gums (arabicetc.) or copolymers thereof, and proteins, including casein, albumin,and keratin. Particles of oligosaccharides, formed of two or moremonosaccharides linked by glycoside bonds, can also be used. Thecopolymer poly (DL-lactide-co-glycolide) is a preferred material, as islatex, an aqueous suspension of a naturally occurring hydrocarbonpolymer. Ribonucleic acids, especially DNA, can also be used to form theparticle component of the particulate antigen complex of the invention.

Suitable organic synthetic polymers include polystyrene, and lower alkylhydrocarbons having between 2 and 6 carbons. The particle component canalso be formed from a metal, especially one of the transition metals ofthe Periodic Table Of Elements, including elements 21-29 (scandiumthrough copper), 39 through 47 (yttrium through silver) and 57-79(lantham through gold) or of oxides of such metals, especially ironoxide.

The materials used to form the particle component can be modified, asnecessary, to include chemical linking groups reactive with the aminoterminus or the carboxy terminus of a protein antigen, or with anyreactive side chain thereof, in accordance with techniques known in theart. Such modified particles, which are typically in the form of beads,are also available commercially. Suitable materials which are availablecommercially include, but are not limited to, DYNABEADS® available fromDynal, Inc. (Great Neck N.Y.) (paramagnetic, polystyrene beads activatedby p-toluenesulfonyl treatment for chemical binding of proteins);BioMag™ iron oxide particles, available for Advanced Magnetics(Cambridge, Mass.); POLYBEAD® microparticles, available fromPolySciences, Inc., (Warrington, Pa.)(monodispersed polystyrene latexbeads and functionalized microparticles); Spherisorb (TM) silica beads,available from Phase Sep.(Norwalk, Conn.). Particles can also bemodified with, an immunoglobulin which in turn can be linked covalentlyto the protein antigen.

Protein, agarose, and polysaccharide-based beads are preferred materialsfor in vivo vaccine applications.

The size of the particle component of the particulate protein antigencomplex has been found to be an important consideration, regardless ofthe size of the protein antigen. In general, average diameter of theparticle component should be at least about 10 nanometers (nm) and nomore than about 50 microns (μm). Preferably, the average diameter of theparticle ranges from about 0.10 μm to about 10.0 μm, and most preferablyabout 0.50 μm to about 6.0 μm.

These materials are provided as examples only and are not intended tolimit the nature of the particle component to be used in accordance withthe present invention. Other materials falling within the specified sizeranges that are not deleterious to the target antigen presenting cell orthe mammalian host can be used, and other suitable materials will bereadily apparent to the skilled artisan.

The protein antigen to be complexed with the particle component can beany protein antigen, especially a protein from a pathogen or tumor cellfor which class I MHC-restricted CTLs appear to be important to conferprotective immunity. As used herein, the term pathogen means anydisease-causing microorganism, including viruses, rickettsia, bacteria,and parasites, especially protozoa and helminths.

In one embodiment, the protein antigen comprises at least oneimmunogenic protein from a pathogen, preferably from a virus or abacterium, which is non-replicating.

When the pathogen is a virus, the protein antigen can be killed orinactivated whole virus; it can be a virus-like particle comprisingenvelope proteins and/or glycoproteins, or it can be protein subunit orfragment thereof from a capsid or envelope antigen or an internalantigen from a virus, in which the virus is a member of familiesincluding adenovirus, picornavirus, corona virus, orthomyxovirus,paramyxovirus, herpes virus, retrovirus or papovavirus. Preferably, theprotein antigen will be from a virus that causes an infection in whichCTLs may play an important role in conferring immunity, such asinfluenza or parainfluenza virus, retroviruses, including HIV-1, HIV-2and SIV, POX viruses (e.g., Varicella zoster), Herpes viruses, includingHerpes simplex 1 and Herpes simplex 2, respiratory syncytial virus,rabies virus, measles virus, polio virus or rotavirus.

Inactivated, non-living whole virus preparations can be prepared inaccordance with any of the techniques known to people skilled in theart, including heat inactivation, and may be used especially inpreparing particulate antigen complexes for polio, influenza, rabies,and Japanese B encephalitis viruses. Virus-like particles arerecombinantly-produced structural proteins that selfassemble intostructures resembling empty virions under defined conditions and arealso potential candidates for vaccine development in accordance with thepresent invention. The preparation and characterization ofrecombinantly-produced virus-like particles have been described forsurface proteins from several viruses, including human papilloma virustype 1 (Hagnesee et al, J. Virol., 67:315 (January 1991); humanpapilloma virus type 16 (Kirnbauer et al., Proc. Natl. Acad. Sci.,89:12180 (December, 1992); HIV-1 (Haffer et al., J. Virol., 64:2653(1990), Hu et al., J. Virol., 179:321 1990); hepatitis A (Winokur, J.Virol., 65:5029 (1991); and human polyoma virus (Rose et al., in press).The teachings of the referenced articles relating to the preparation,characterization, and purification of virus-like particles are herebyincorporated by reference. These virus-like particles, which resemblelive virus in external conformation but are non-infectious, may beprocessed and presented by class I MHC molecules of antigen presentingcells in a manner analogous to that for live virus and are goodcandidates for vaccine development.

For many viruses, one or more individual antigens may be predominantlyimportant for conferring immunity, including a CTL component, so thatvaccines can be comprised of that protein subunit, or immunogenicfragment thereof. One example is the surface antigen of the hepatitis Bvirus, HBSag, that is secreted from cells and present in the blood ofinfected human beings. A second example is the influenza hemagglutinin(HA) antigen, which can be chemically removed from the virus capsid orproduced recombinantly using techniques that are old and well known inthe art.

Protein subunits and fragments can be obtained by conventionaltechniques, such as proteolysis, chemical treatment, or solubilizationand purification of the relevant protein from the native virus, they canbe prepared using automated peptide synthesis techniques, or they can beproduced by recombinant DNA techniques and then purified in accordancewith procedures known to persons skilled in the art. When the antigen isobtained through recombinant DNA techniques, DNA including that encodingthe antigen of interest is cloned into an expression vector, such as avaccinia virus, baculovirus, plasmid, or phage and expressed in asuitable prokaryotic or eukaryotic expression system, in accordance withestablished protocols. See, Sambrook, Fitsch, & Maniatis, MolecularCloning, Chapters 8 and 9 (second edition, 1989), which are herebyincorporated by reference.

When the protein antigen is from a bacterium, the antigen can be frominactivated bacteria, it can be from a toxin or a capsularpolysaccharide, or it can be a subunit antigen. Preferably, the proteinantigen is from a bacteria where cellular immunity appears to beimportant for providing protection against infection or reinfection, forexample bacteria causing tuberculosis (Mycobacteria tuberculosis);leprosy (Mycobacterium leprae), brucellosis (Brucella spp.) andlisteriosis (Lysteria monocytogenes). Such protein antigens can beprepared readily by persons skilled in the art, by traditional orrecombinant DNA techniques.

In yet another embodiment of the invention, the non-replicating proteinantigen can be from a parasite where CTL response appears to beimportant for conferring immunity. Such parasites include members fromthe class Apicomplexa, which includes Plasmodium species that are theetiologic agents of infectious malaria and Toxoplasmosis gondii, theetiologic agent of toxoplasmosis. Also included are protein antigensfrom Leischmania species.

Tumor-associated antigens, i.e., antigens associated with neoplasticdisease, can also be used as the antigen in the particulate antigencomplexes of the invention. Preferably, the tumor antigen will be anantigen associated with human melanoma, colonic carcinoma, breastcarcinoma, or renal carcinoma. It has previously been shown, forexample, the autologous CTLs recognize a total of six independentantigens on human melanoma cells, which can be used in the particulateprotein antigen complexes of the invention. See, Van Der Bruggen, etal., Science, 254:1643 (December, 1991).

The linkage between the antigen and the particle of the invention can beformed in one of a variety of ways. What is important is that thelinkage is sufficient to physically associate the two components of thecomplex. The linkage to be used for a given complex will depend upon thecomposition of the particle and the protein antigen. For some complexesthe linkage will best be made by chemical means (covalent linkages),while for other particle complexes, linkage through non-covalentassociations, such as adsorption of the protein antigen to the particlecomponent, will suffice.

The elements of the complex can be covalently linked by means well knownto persons skilled in the art, including standard dehydration reactionsusing carbimides or by a large variety of heterobifunctional agents orlinkers. Covalent linkages through peptide bonding of the amino orcarboxy terminus of the protein antigen or any appropriate amino acid inthe side chain thereof with reactive groups on the particle component isa particularly preferred linkage. Particularly useful linkages alsoinclude those which generate a disulfide link at one functional groupand a peptide link at the other, includingN-succidimidyl-3-(2-pyridyldithio) propionate (SPDP) (Pierce Chemicals)(Rockford, Ill.). This reagent creates a disulfide linkage betweenitself and a cysteine residue in one protein and an amide linkagethrough the amino group on a lysine or other free amino group on theother component. A large number of disulfide/amide forming agents areknown. Other bifunctional linking agents form a thioester rather than adisulfide linkage. The ability to make such linkages is well within theskill in the art, and the skilled artisan can readily identify asuitable linkage for a given complex.

The particulate antigen complexes of the invention are useful in vaccinedevelopment with non-living vaccines and are also useful in vaccinatingmammalian hosts for CTL immunity. The complexes of the invention mayalso be useful in the treatment of infectious diseases, such as HIV-1and HIV-2, where CTL response to infection may play an important role.

For example, the particulate protein complexes of the invention can beused in CTL vaccine development. In one embodiment, the complexes aremade as described herein and evaluated for their activity in an in vitroantigen presenting assay similar to the one described in the Examples.The identification method involves obtaining a plurality of potentiallyimmunogenic antigens from a pathogen, such as a virus, for which a CTLvaccine is sought, linking the protein antigens to a particle to formparticulate antigen complexes; adding the particulate antigen complexesto a population of antigen presenting cells, including macrophages, andlymphokine secreting CTLs or antigen-specific T--T hybridomas thatpreviously have been stimulated with an appropriate antigen, such as awhole virus; and selecting a complex including a protein antigenrecognized by the previously stimulated CTLs. This in vitro assay systemenables the identification of active complexes and can also optimizeidentification of the component of the pathogen recognized by CTLs. Theantigen presenting cells used in the antigen presentation assay mustinclude a subpopulation of macrophages that have the ability to take up,process, and present antigen in association with class I MHC. Inaccordance with the present invention, it has been discovered that mousebone marrow cells, thymic macrophages, spleen cells, and resident andstimulated peritoneal macrophages all include a subpopulation ofmacrophages exhibiting this activity, and can therefore be used in thein vitro assay of the invention.

One of several antigen presentation assays known in the art can be usedin this in vitro screen. Preferred assays will employ a read out thatmeasures lymphokine or serine esterase production by CTLs or by classI-restricted T--T hybridomas. Alternatively, if the subpopulation ofmacrophages that have the ability to present exogenous antigen inassociation with class I can be lysed by CTLs, the read out system canemploy a standard chromium release assay well known to those of skill inthe art. The preparation of class I restricted T--T hybridomas using aBW5147 cell line transfected to express the CD8 gene has been describedin the literature (Rock et al., J. Immunol., 145:804 (1990), theteachings of which are hereby incorporated by reference. Thesetechniques can be used to prepare antigen-specific, class I-restrictedT--T hybridomas useful in the screening assays of the invention.Particulate antigens that elicit a strong CTL response in vitro are goodcandidates for vaccine development for infectious diseases ortumor-associated pathological disorders where the cellular arm of theimmune response may be important to confer complete protection.

In another embodiment relating specifically to vaccine development,candidate complexes can also be tested in a suitable animal model, todetermine whether a candidate vaccine comprising a particulate proteinantigen complex of the invention can confer immunity against infectionwith the live pathogen or whole tumor cells. The ability of a givenparticulate antigen complex to confer protective immunity againstinfectious disease in a animal can be established using challengeassays, such as lethal or sub-lethal challenge assays, known in the art.For example, where the animal model is a laboratory animal such as amouse or a rat and the antigen is a viral antigen, the animal isimmunized with the candidate particulate antigen complex of theinvention via a suitable route of administration, such assubcutaneously, intravenously or intraperitoneally, with or withoutboosting, and subsequently challenged with lethal doses of virus in asuitable carrier. Survival of the immunized animals is monitored andcompared to virus-immune positive control and negative control animals,with have been immunized with live virus and soluble antigen,respectively. A lethal challenge assay that can be used is described,for example, in Dillon et al, Vaccine, 10:309 (19912).

In another embodiment, the particulate antigen complexes of theinvention are used in pharmaceutical compositions that, whenadministered to a mammalian host in an effective amount, are capable ofinducing CTL immunity. In accordance with the present invention, theparticulate antigen complexes will have utility in both medical andveterinary applications and can be used therapeutically, as well asprophylactically. The term mammal as used herein includes both human andnon-human primates, including chimpanzees and monkeys, and domesticatedmammals, such as dogs, cats, rabbits, guinea pigs, pigs, cows, horses,sheep, and goats, as well as common laboratory animals such as mice,rats, and hamsters.

The pharmaceutical compositions of the invention comprise apharmaceutically acceptable excipient, at least one immunogenicparticulate antigen complex of the invention, in which the particulateantigen complex comprises a particle having an average diameter of about10 nm to about 50 μm and non-replicating protein antigen, and optionallyother ingredients, described supra. The excipient must bepharmaceutically acceptable, in the sense of being compatible with theactive ingredient(s) of the pharmaceutical composition and notdeleterious to the recipient thereof. Examples of suitable excipientsare, for example, water, saline, dextrose, glycerol, ethanol or thelike, and combinations thereof. In addition, if desired, thepharmaceutical composition may contain minor amounts of auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,bacteriostat or adjuvants which further enhance effectiveness of thevaccine.

The preparation of such pharmaceutical compositions is well understoodin the art. Typically, they are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in orsuspension in liquid prior to injection. The preparation may also beemulsified. The compositions may also be formed for oral delivery, inwhich case the formulation will normally contain such excipient as, forexample, pharmaceutical grades of mannitol, starch, lactose, magnesiumstearate, sodium saccharide, cellulose, magnesium carbonate and thelike. These compositions can take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, and powders.

The compositions of the invention are administrated to a mammal in needof such treatment, such as a mammal at risk of infection from orinfected with a pathogenic organism or at risk of developing atumor-related disorder, in a CTL-stimulatory amount. Administration ofthe pharmaceutical compositions of the invention may be by any suitableroute including oral, nasal, topical and parenteral, with oral andparenteral routes, including subcutaneous, intramuscular, intravenous,and intradermal, being preferred.

By CTL-stimulatory amount is meant that the pharmaceutical compositioncontains sufficient amount of the particulate protein antigen complex ofthe invention to induce CTL response to the subject antigen. The preciseamount to be administered will vary depending upon the protein antigen,mode of administration selected, and the mammalian host to be immunized.

The invention is more fully described in the following Examples. TheseExamples are provided for illustrative purposes only and are not meantto limit the invention in any way.

EXAMPLE 1

Exogenous antigens in the extracellular fluids do not gain access to theclass I antigen presenting pathway in most cells. However, there is anAPC resident in spleen that can process and present exogenous Ags inassociation with class I molecules. In this Example, we characterize thephenotype of this cell. This APC is of low buoyant density, is adherentto sepharose and glass, and expresses both class II molecules andFc-receptors. This phenotype identifies this APC as a macrophage.Resident, peptone- and thioglycolate-induced peritoneal macrophages alsodisplay this antigen presenting activity. Analysis with CTL clonessuggest that this antigen presenting pathway may be active in only asubset of macrophages. A similar antigen presenting activity is alsopresent in dendritic cell-enriched populations from spleen although wecannot rule out the possible involvement of contaminating macrophages.In contrast, B and T cells that are resident in spleen and LPS blastsare unable to present exogenous Ags in association with class Imolecules.

Methods And Materials

Mice

C57BL/6 mice, ages 5-8 weeks were purchased from the Jackson Laboratory(Bar Harbor, Me.) or were bred at the Dana-Farber Cancer Institute.

Reagents

Chicken OVA was purchased from ICN Immunobiologicals, Lisle, Ill., orSigma, St Louis Mo. Isotonic BSA (pathocyte 4) was purchased from ICNImmunobiologicals, Lisle, Ill.

Cell Lines

The RF33.70 (anti-OVA+K^(b)) (14) T cell hybrid has been previouslydescribed.

Cell Fractionations and Incubations

Splenocytes were depleted of erythrocytes by treatment with Tris-NH₄ Cland depleted of accessory cells by passage over two successive G10sephadex columns (Ly et al., Immunol. Methods, 5:239 (1974)). B cellswere positively selected by panning on rabbit anti-Ig coated dishes.LPS-blasts were prepared by incubating G10-passed splenocytes with LPS(10 μg/ml) for 72 hrs at 37° C. Splenocytes were fractionated into low(floating above 25% isotonic BSA) and high density fractions bycentrifugation on BSA gradients, essentially as previously described(Beller and Unanue, J. Immunol., 118:1780 (1977); Grant and Rock, J.Immunol., 148:13 (1992). In some experiments, low density splenocyteswere incubated on sterile petri dishes at 37° C. and glass-nonadherentcells collected after 2 hrs and the remaining cells incubated foranother 18-24 hrs; nonadherent cells were collected and adherent cellswere harvested at the later time points by scraping. In some experimentslow density cells, or 24 hr glass-nonadherent low density cells werefurther fractionated by rosetting with sheep red blood cells (SRBCs)sensitized with rabbit anti-SRBC Ab (kindly provided by Dr. G. Sunshine,Tufts University, Boston, Mass.), as previously described (Steinman etal. J. Exp. Med., 149L:1 (1979); Sunshine et al., J. Exp. Med., 152:1817(1980)). Peritoneal exudates were harvested by lavage from naive mice ormice injected three days earlier with approximately 1.5 mls ofthioglycolate or peptone broth. In some cases, peritoneal exudate cellswere cultured for 18-24 hrs on flat bottom plastic microtiter wells andthen separated into adherent and nonadherent fractions.

Cell Culture

Media was RPMI 1640 (Irvine Scientific, Santa Ana, Calif.) supplementedas previously described (Rock et al, J. Immunol., 145:804). T--Thybridoma cultures were prepared as previously described (Rock et al.,J. Immunol., 145:80414). Briefly, microcultures were prepared with5-10×10⁴ T--T hybrids in the presence or absence of APCs, and with/orwithout antigen in 200 ml media in flat bottom microtiter wells induplicate. The precise culture constituents are detailed in therespective experimental protocols. Dose response curves were generatedby titrating the number of APCs per well in the presence or absence of aconstant amount of Ag, or by titrating the concentration of Ag with aconstant number of APCs per well. After 18-24 hours incubation at 37°C., a 100 ml aliquot of supernatant was removed and subjected tofreeze-thawing.

Mitogen responsiveness was determined by incubating splenocytes (2×10⁵cells/well) with or without Con A (5/ml) or LPS (10 m/ml) in flat bottommicrotiter plates in triplicate. After 48 hrs incubation at 37° C., theincorporation of ³ H-thymidine into DNA was determined.

Lymphokine and Cytotoxicity Assays

IL-2 content in T cell hybridoma culture supernatants was measured witha quantitative bioassay with HT-2 cells (Watson, J. Exp. Med., 150:1510(1979)) as previously described (Kappler et al., J. Exp. Med., 153:1198(1981); Rock and Benacerraf, J. Exp. Med., 157:1618 (1983)). In theabsence of antigenic stimulation the CPM was usually <1×10³.Cell-mediated cytotoxicity was measured using ⁵¹ Cr release assays aspreviously described (Rock, J. Immunol., 145:804)

Results

Accessory cell-depletion removes APCs that process and present exogenousAgs with class I molecules.

In our initial studies, we demonstrated that the splenic APCs thatprocess and present exogenous Ags with class I molecules expressed classII molecules. (Rock, K. L., S. Gamble, and L. Rothstein. 1990 Science249:918). In spleen, class II molecules are expressed on B lymphocytes,dendritic cells and macrophages. The latter cells bind to and can beefficiently removed by incubation with G10 sephadex. (Ly, I. A., and R.I. Mishell. 1974. J. Immunol. Meth. 5: 239). In this experiment, wedemonstrated that accessory cell depletion removes APC that process andpresent exogenous antigen with class I molecules.

Cultures were prepared with RF33.70 T cell hybrids (anti-OVA+K^(b), 10⁵/well), APCs (splenocytes) (10⁶ /well) and with or without theconcentration of OVA indicated in FIG. 1A, in flat bottom microtiterwells (200 ml) in duplicate, as described in the Methods And Materials.Unfractionated splenocytes (closed circles in FIG. 1A), G10-passedsplenocytes (closed triangles) or sIg+ B cells positively selected fromG10-passed spleen by panning on anti-Ig-coated plates (open circles)were used as a source of APCs. After 18 hrs incubation at 37° C., analiquot (100 μl) of culture supernatant was removed, freeze-thawed, andassayed for IL-2 content with HT-2 cells. Data are expressed as the meanCPM of ³ H-thymidine incorporated by HT-2 cells. As a control for thefunctional integrity of the T and B lymphocytes in the fractionated cellpopulations, these cells (2×10⁵ /well) were cultured with Con A (5mg/ml) or LPS (10 mg/ml) in flat bottom microtiter wells (200 ml) intriplicate. After 48 hrs incubation the mean CPM of ³ H-thymidine×10⁻³±SEM incorporated for unfractionated, G10-passed and G10->sIg⁺ cellsstimulated with Con A was 274±9.2, 260±8.1 and 7.4±0.8, respectively,and stimulated with LPS was 387±13.4; 416±14.4; 345±19.2, respectively;the CPMs without mitogen were <3×10⁻³.

As shown in FIG. 1A, passage of splenocytes over a C10 column markedlydecreased the ability of these cells to present exogenously-added OVA tothe OVA+K^(b) -specific T cell hybrid, RF33.70. The percentages of B(sIg⁺) and T (Thy-1⁺) cells were not appreciably altered by passage oversephadex as assessed by immunofluorescence and flow fluorocytometry(data not shown). Furthermore, the B cells in the G10-fractionatedspleen appeared to be functionally competent, as their responses to LPSstimulation were similar to controls, as expected. Similar results wereobtained when sIg+ cells from G10-passed spleen and positively selectedby panning (FIG. 1A). These results suggest that the majority of Blymphocytes in spleen are incapable of presenting exogenous OVA inassociation with class I molecules. This conclusion is further supportedby experiments with density-fractionated APCs that are described below.

The exogenous Ag-class I APC in spleen is enriched in M.Oslashed./dendritic cell populations.

Macrophages (M.O slashed.) and dendritic cells have low buoyant densityand can be enriched by isopycnic centrifugation. (Beller, D. I., and E.R. Unanue. 1977 J. Immunol. 118:1780; Steinman, R. M., G. Kaplan, M. D.Witmer, and Z. A. Cohn. 1979 J. Exc. Med. 149:1). In this experiment, wedemonstrated that splenic APCs that present exogenous OVA in associationwith class I MHC molecules are greatly enriched in the low densityfractions of spleen. See FIG. 1B.

Splenocytes were fractionated by centrifugation on discontinuous BSAdensity gradients, as described in the Materials and Methods section. Inthis experiment, 1.4% of the splenocytes were recovered in the lowdensity fraction. Cultures were prepared as described above andcontained RF33.70 T cell hybrids (anti-OVA+K^(b), 10⁵ /well), ±OVA (2mg/ml) and, as a source of APCs, the number of unfractionated (closedcircle), high density (closed triangle) or low density (open triangle)splenocytes, as indicated in FIG. 1B. Cultures were otherwise preparedand handled as described above.

One and one half to five percent of splenocytes were recovered in thelow density fraction and, as illustrated in FIG. 1B, there was acorresponding 20 to 40-fold enrichment in the APC activity. Most Blymphocytes and virtually all T lymphocytes are present in the higherdensity fractions of spleen.

Dendritic cells and macrophages differ in the expression of Fc-receptorsand the Fc-receptor has been used to fractionate these two cellpopulations through resetting with antibody (Ab) -coated erythrocytes.Therefore, we incubated the low density fraction of spleen withAb-coated erythrocytes and examined the activity of resetting andnonrosetting populations.

Briefly, splenocytes were fractionated by centrifugation on BSA densitygradients and 3.8% of cells were recovered in the low density fraction.This fraction is represented by the open triangles in FIG. 1C. The lowdensity cells were then incubated with SRBCs sensitized with rabbitanti-SRBC Ab and the resetting (FCR+, open circle) and non-rosetting(FCR-, closed triangle) cells separated by centrifugation onficoll-hypaque gradients. The erythrocytes were subsequently lysed bytreatment with tris-NH₄ Cl. Cell recoveries were: 47% resetting withIg-sensitized SRBCs and 53% non-rosetting; 4.6% of LDCs rosetted withunsensitized SRBCs. The APC activity of the various fractionated andunfractionated cell populations was assayed in cultures prepared andassayed as described above.

As shown in FIG. 1C, the antigen presenting cells capable of presentingsoluble antigen in association with class I MHC (sometimes hereinafterreferred to as "ExAPC/I") were positively-selected by binding toAb-sensitized erythrocytes. Approximately 50% of the low density cellswere recovered in this resetting fraction. This fractionation wasdependent on the SRBC-bound Ab since few cells (<5%) were recovered whenlow density cells wee rosetted with unsensitized erythrocytes. We alsofound reduced, but detectable, ExAPC/I activity in the nonrosetting APCpopulation.

We next examined the adherence properties of the APCs in the low densityfractions of spleens that present OVA in connection with class I MHC.

Low density splenocytes were prepared as described above and an aliquotwas incubated on glass petri dishes. After incubation for 2 hrs at 37°C., the nonadherent cells were collected. The 2 hr adherent cells werecultured for another 22 hrs and then fractionated into nonadherent andadherent fractions. The APC activity of the various fractionated andunfractionated cell populations was assayed with RF33.70 cells, ±OVA (4mg/ml) in cultures prepared and assayed as described, except that theunfractionated LDC and 2 hr nonadherent LDC were added to the microtiterplates and incubated for 24 hrs (in parallel to the group incubated onglass for 24 hrs) before the addition of the T--T hybrids and Ag; usingthis experimental design all APC population were cultured for the samelength of time.

As shown in FIG. 2, the ExAPC/I present in the low density fractions ofspleen were adherent to glass (2 hr incubation); when the 2 hrglass-adherent cells were cultured for 24 hrs, the APC activity could berecovered in both released and adherent populations. See FIG. 2. Thelatter population is enriched for macrophages. The cells that detachfrom glass during the 24 hour incubation have been previously reportedto be composed of both dendritic cells and macrophages. (Steinman, R.M., G. Kaplan, M. D. Witmer, and Z. A. Cohn. 1979 J. Exp. Med. 149:1;Sunshine, G. H., D. R. Katz, and M. Feldmann 1980. J. Exp. Med.152:1817).

The adherence properties and expression of FcR strongly suggest that atleast some M.O slashed.s can present exogenous Ags in association withclass I. This conclusion is supported by analyses of peritonealmacrophages described below. The finding that this APC activity issomewhat enriched in cell fractions that should be enriched in dendriticcells (FIG. 2) may indicate that cultured dendritic cells alsoparticipate in this response, however, we cannot rule out the potentialcontribution of contaminating M.O slashed.s.

Activated B cells do not present exogenous Ag in association with classI MHC molecules.

Activation of B lymphocytes increases their ability to present antigensto class II MHC-restricted T cells (Chesnut, P. W., and H. W. Grey.1986, Adv. Immunol. 39: 51). To directly examine the antigen presentingcell activity of activated B cells, we stimulated G10-passed splenocyteswith lipopolysaccharide (LPS), as described below.

G10-passed splenocytes were cultured with LPS (10 μg/ml). After 72 hrsincubation at 37° C., lymphoblasts were isolated by centrifugation onficoll-hypaque. The APC activity of the LPS blasts (closed circle inFIG. 1D) or thioglycolate-induced PECs (open circle in FIG. 1D) wasassayed in cultures with RF33.70 cells and OVA (4 mg/ml) as described.As a positive control, PEC APCs were added to parallel cultures andpresented with exogenous OVA to RF33.70.

As shown in FIG. 1D, LPS blasts failed to present exogenous OVA toRF33.70.

Peritoneal macrophages can also present exogenous Ag in association withclass I MHC molecules.

Macrophages are normally present in the peritoneal cavity and can berecruited by the injection of thioglycolate and peptone into this site.We examined the activity of the cells found in the peritoneum underthese conditions. The results are illustrated in FIGS. 3A and 3B.

In this experiment, cells were obtained by peritoneal lavage from naivemice or mice injected three days earlier with thioglycolate or peptonebroth. The APC activity of these PECs and unfractionated splenocytes wasassayed in cultures with RF33.70 and OVA (4 mg/ml) that were preparedand assayed as described. Thioglycolate-induced PECs were incubated onplastic microtiter wells for 18 hrs at 37° C. after which time thegroups indicated in FIG. 3A were separated into adherent and nonadherentcells. The APC activity of these cells was then assayed in cultures withRF33.70 cells and the indicated titration of OVA as described forFIG. 1. The CPM from groups without OVA was <1300.

As shown in FIGS. 3A and 3B, the resident cells in the peritoneal cavitycan present exogenous OVA to RF33.70 cells. Peritoneal exudates, inducedwith either thioglycolate or peptone were enriched in cells with thisactivity, as shown in FIG. 3A. These APCs were adherent to plastic (FIG.3B). The plastic-nonadherent APC fractions were less active in thisassay system. These findings lend further support to the conclusion thatM.O slashed.s can present exogenous Ags in association with class I andindicate that this property is not limited to APCs resident in spleen.

EXAMPLE 2

In this experiment, we explored whether antigen could be moreefficiently targeted to the subpopulation of antigen presenting cellsdescribed in Example 1 for uptake and entry into the class I pathway bypreparing several different complexes of the OVA protein and assayingthe ability of the complexes to be presented by class I by differentAPCs, as described below.

Experiment A.

In this experiment, chicken OVA was covalently coupled with iron oxideparticles to form a particulate antigen complex, and APC presentation ofthe complex by class I MHC molecules was compared to that foruncomplexed, soluble OVA. The chicken OVA, purchased from ICNImmunobiologicals, was covalently coupled to BIOMAG (TM) magnetized ironoxide particles which are coated to provide amino groups (averagediameter, 1 μm)(Advanced Magnetics, Cambridge, Mass.) in accordance withthe manufacturer's instructions for attaching proteins usingglutaraldehyde as the coupling agent. See also, Weston & Avrameas,Biochem. Biophys. Res. Comm., 45:1574 (1971). Briefly, The BioMagsuspension of iron oxide particles was first activated withglutaraldehyde. 10 ml of BioMag was transferred to a flat-bottomreaction flask which comfortably holds 50 mL. The coupling buffer (0.01Mpyridine) was added to a volume of about 50 ml, the flask was shakenvigorously, after which the BioMag particles were separatedmagnetically. The liquid in the flask was then aspirated, leaving theBioMag as a wet cake on the container wall. The washing procedure wasthen repeated with three more additions with the coupling buffer, withthe contents of the flask being shaken well after each addition of thebuffer.

After the final aspiration, 20 ml of 5% glutaraldehyde was added to thewet BioMag cake, the flask was shaken vigorously, and the flask thenagitated at room temperature for three hours. After three hours, theBioMag was magnetically separated and the unreacted glutaraldehyderemoved by aspiration. 50 ml of coupling buffer was then added to theflask and the flask was vigorously shaken. The procedure was repeated atotal of four times, and the final volume of coupling buffer wasaspirated, leaving the activated BioMag as a moist cake on the sides ofthe flask.

Following activation, 10-18 mg of OVA was added to 10 ml of couplingbuffer and added to the BioMag cake and the mixture shaken vigorously.The flask was then agitated overnight at room temperature and thefollowing day the OVA/BioMag complexes were magnetically separated andthe contents of the flask aspirated. 50 ml of glycine quenching solutionwas then added and the contents of the flask were shaken vigorously.After quenching, approximately 50 ml of coupling buffer was added to theBioMag cake, the flask shaken vigorously, and the contents separatedmagnetically. This washing procedure was repeated four times.

Separate cultures were then prepared with RF33.70 T--T hybridomas (10⁵hybrids per well), as described in Example 1, various APCs (10⁵ APCs perwell) and with varying concentrations OVA or OVA-iron oxide particle, asindicated in FIG. 4A, in flat bottom microtiter wells (200 μl) induplicate. Antigen presenting cells were A3.1, (a macrophage clonederived by retroviral immortalization of murine bone marrow cells,followed by selection of an immortalized clone that exhibited thephenotype and antigen presenting capability of the above-describedantigen presenting cells) and EL4, a readily available T cell tumorwhich, like most cells, is unable to present exogenous antigen withclass I MHC molecules.

Retroviral immortalization of bone marrow macrophages was achievedsubstantially as described in Blasi et al., Nature, 318:667 (December1985), which is hereby incorporated by reference.

After incubation for 18 hours, 37° C., an aliquot (100 μl) was removedfrom each well and assayed for IL-2 content with HT-2 cells, aspreviously described.

The results of the experiment are illustrated in FIG. 4A, in which thedata represent the mean counts per minute (CPM) of tritiated thymidineincorporated by HT-2 cells (5×10³) for the duplicate cultures. Asillustrated in the Figure, the OVA-iron oxide particle complex (closedcircles in FIG. 4A) was presented almost 10,000 fold more efficientlythat soluble antigen (open circles) by the macrophage clone, A3.1. EL4cells, which are not phagocytic and do not present exogenous antigenwith class I, were unable to present the particulate protein antigen orthe soluble antigen (open squares in FIG. 4A).

Experiment B.

This experiment was similar to the one described in Experiment 2A above,except that, in addition to linking OVA to the iron oxide beads, OVA wasalso linked to a different particle type, a silica bead, having anaverage diameter of about 5 μm.

OVA protein was linked to 5 μm silica beads, available from PhaseSeparation (Norwalk, Conn.) by mixing the protein antigen and the 5 μmbeads, in accordance with the manufacturer's instructions. Briefly, thesilica beads and OVA protein were mixed in 0.5% deoxy chololate (DOC) in10 mM Tris-phosphate buffered saline (TBS) and dialyzed at 4° C. forapproximately 36 hours to remove the detergent. Ratios used were 600 μgprotein per 10⁷ beads and 5 nm lipid per 10⁷ beads with 2 to 20×10⁶beads. Dialysis was against the 0.6 L TBS in a sterile tissue cultureflask containing SM-2 biobeads (BioRad, Richmond, Calif.) prepared inaccordance with the manufacturer's instructions and used 1 gm per ml ofsample being dialyzed, as a detergent absorbent. The flask was placed onan agitating platform to keep the beads in suspension during dialysis.After 24 hours of dialysis, 5 mM lf CaCl₂ was added to the dialysisbuffer. Dialysis tubing was treated in boiling water and closed withclips after sample addition to exclude air.

After dialysis, the dialysis bag was cut open, and the beads wereremoved and washed several times in sterile medium and then stored at 4°C. until added to the culture.

Cultures with the particulate-OVA complexes were then prepared asdescribed above and, after 18 hours incubation under the conditionsdescribed, supernatants were removed and assayed for IL-2 activity, alsoas described.

The results of this experiment are set forth in FIG. 4B, in which theopen circles represent the activity of soluble OVA in the antigenpresenting assay; the closed triangles represent the activity of theOVA-silica complex; and the closed circles, the activity of the OVA-ironoxide complex. These results show that a different type of particle wasactive in the particulate antigen complex in inducing CTL response. Asillustrated in the Figure, the OVA-iron oxide particle complex was onceagain presented almost 10,000 fold more efficiently than soluble antigenby the macrophage clone, A3.1; the OVA-silica complex was also presentedwith much greater efficiency.

Experiment C.

This experiment was similar to the one described in Experiments A and Babove, except that the OVA was adsorbed to polystyrene microspheres.

OVA was covalently linked to POLYBEAD (R) polystyrene microspheres(average diameter 3 μm; 2.5% solids--latex) (Polysciences, Inc.Warrington, Pa.), in accordance with the manufacturer's instructions.Briefly, the POLYBEADS were suspended and washed twice in buffer (0.1Mborate, pH 8.5), spun down, and then resuspended two times.

2-3 mg/ml of OVA was added to the mixture and the mixture was agitatedovernight. The following day, the beads were collected by centrifugationand washed twice in buffer containing 0.1% BSA.

Cultures were then prepared as described above and, after 18 hoursincubation under the conditions described, supernatants were removed andassayed for CTL activity, also as described.

The results of this experiment are set forth in FIG. 4C, in which theopen circles represent the activity of soluble OVA in the antigenpresenting assay and the closed circles represent the activity of theOVA-latex complex. These results show that a different type of particleand linkage were active in the particulate antigen complex in inducingCTL response. As illustrated in the Figure, the OVA-latex particulateantigen complex was more efficiently presented by the macrophage clone,A3.1 than soluble OVA.

EXAMPLE 3

This Example demonstrates that particulate antigen is presented bynormal antigen presenting cells with much greater efficiency thansoluble antigen.

The Experiments described in Example 2 were all conducted with the A3.1immortalized macrophage clone. We therefore wished to determine whethernormal antigen presenting cells would also exhibit greater efficiency inpresenting particulate antigen complexes with class I MHC molecules.

In this Example, the antigen presenting cells were peritoneal exudatecells (PECs) obtained from mice after intraperitoneal injection withabout 1.5 mls of thioglycolate media. Peritoneal exudates were harvestedby lavage after 72 hours and used in and assay performed substantiallyin accordance with Example 2A.

Chicken OVA was coupled with BIOMAG iron oxide particles as described inExample 2, to form a particulate antigen complex, and APC presentationof the complex by class I MHC molecules was compared to that foruncomplexed, soluble OVA. Briefly, chicken OVA, (ICN Immunobiologicals),was covalently coupled to BIOMAG (TM) magnetized iron oxide particles inaccordance with the manufacturer's instructions for attaching proteins.Separate cultures were then prepared with RF33.70 T--T hybridomas (10⁵hybrids per well), as previously described, PEC APCs (10⁵ APCs per well)and with varying concentrations OVA or OVA-iron oxide particle, asindicated in FIG. 5, in flat bottom microtiter wells (200 μl) induplicate.

After incubation for 18 hours, 37° C., an aliquot (100 μl) was removedfrom each well and assayed for IL-2 content with HT-2 cells, aspreviously described.

The results of the experiment are illustrated in FIG. 5, in which thedata represent the mean counts per minute (CPM) of tritiated thymidineincorporated by HT-2 cells (5×10³) for the duplicate cultures. Opencircles represent the activity of soluble OVA in the antigen presentingassay; closed circles represent the OVA-iron oxide complex. Asillustrated in the Figure, the OVA-iron oxide particle complex waspresented by the PEC APCs much more efficiently than soluble OVA.

EXAMPLE 4

This Example demonstrates the ability of particulate whole proteinantigen complexes to prime antigen-specific CTL responses in vivo.

Experiment A.

OVA-iron oxide particles and soluble OVA were prepared as described inExample 2A, formulated with phosphate buffered saline (PBS), andseparately injected subcutaneously into both flanks of C57Bl/6 mice,purchased from the Jackson Laboratory, Bar Harbor, Me., or bred at theDana-Farber Cancer Institute (Boston, Mass.). The amount of proteinantigen complex injected was either 90 μg or 18 μg.

Seven days following the injections, the mice were sacrificed and theirspleen cells removed. Spleen cells (30×10⁶) were restimulated in vitrofor five days with X-irradiated (20,000 rads) EG7-OVA cells (15×10⁶cells), in 10 ml of media. EG7 is a EL4 tumor cell line transfected withOVA cDNA)(Moore et al, Cell, 54:777 (1988)).

After five days of culture, the cells were tested for their ability tolyse ⁵¹ Cr-labeled EL4 cells (C57BL/6, H2^(b) thymoma) or EG7 cells, inaccordance with conventional techniques.

The results of the experiment are illustrated in FIGS. 6A-6D. As shown,the particulate OVA antigen complexes primed CTL response at 90 and 18μg (FIGS. 6A and 6B, respectively); the same amount of soluble OVA wasineffective (FIGS. 6C and 6D, respectively) to elicit a CTL response.

Experiment B.

This experiment was similar to the one described in paragraph Aimmediately above, except that the antigen used was native E. coliβ-galactosidase (β-gal) (mw approximately 540 kd), instead of chickenOVA. β-gal-iron oxide particles were prepared as described in Example2A, formulated with phosphate buffered saline (PBS) and separatelyinjected subcutaneously into both flanks of Balb/c mice. The amount ofantigen injected was either 13 μg or 130 μg, as indicated in FIGS. 7Athrough 7C. Five weeks following the injections, the mice weresacrificed and their spleen cells removed. Spleen cells (35×10⁶ wererestimulated in vitro for five days with 3×10⁶ X-irradiated (20,000rads) P13.4 cells. P13.4 is a mastocytoma cell line transfected withβ-gal cDNA, that was generated in accordance with establishedrecombinant DNA techniques.

After five days of culture, the cells were tested for their ability tolyse ⁵¹ Cr-labeled P815 cells (DBA/2, H2^(d) mastocytoma) or P13.4cells, in accordance with conventional techniques.

The results of the experiment are illustrated in FIGS. 7A-7C. As shown,the particulate β-gal antigen complexes primed CTL response at 13 μg(FIG. 7A), while the same amount of soluble β-gal (FIG. 7B), and tentimes the amount of β-gal (FIG. 7C), were not effective.

Experiment C.

This Experiment was similar to the ones described in the precedingparagraphs, except that the antigen used was native hen egg lysozyme("HEL") (mw approximately 15 kd). HEL-iron oxide particles were preparedas described in Example 2A, formulated with phosphate buffered salineand (PBS) injected subcutaneously into both flanks of C57Bl/6 mice.Three weeks following the injections, the mice were sacrificed and theirspleen cells removed. Spleen cells (5×10⁶) were restimulated in vitroovernight with 5×10⁶ X-irradiated (20,000 rads) a CNBr-cleaved peptidepreparation of HEL (100-300 μg/ml), in 1 ml media with rat con ASN/alpha methyl mannoside (05. %).

After five days of culture, the cells were tested for their ability tolyse ⁵¹ Cr-labeled EL4 cells in the presence of CNBr-cleaved peptides ofHEL or EL4 cells, in accordance with conventional techniques.

The results of the experiment are illustrated in FIGS. 8A and 8B. Asshown in the Figure, the particulate HEL antigen complexes primed CTLresponse (FIG. 8A), while the naive (unprimed) animals did not respondunder the same conditions.

The results of the foregoing experiments, which were conducted withunrelated antigens, establish a general pathway for uptake, processing,and presentation of antigen by a subpopulation of antigen presentingcells.

EXAMPLE 5

This Example describes the induction of a CTL response to an HIV antigenin mice in vivo using a particulate antigen complex of the invention.

Soluble recombinant HIV-1-IIIB gp160 envelope glycoprotein is preparedfrom cells infected with a recombinant baculovirus expressing the genefor gp160 of HIV-1-IIIB, as previously described. Javaherian et al.,Proc. Natl. Acad. Sci. USA, 86:6768 (1989). The protein can be purifiedas also described by Javaherian et al. The purified product is a singleband on SDS-PAGE, as seen by both Coomassie blue staining and by Westernblotting with a monoclonal anti-gp41 antibody.

The purified HIV-1 gp160 envelope protein is then linked to POLYBEADAmino Microspheres (Polysciences, Inc) (1 μm average diameter) usingglutaraldehyde to activate the particles, in accordance with themanufacturer's instructions. The particulate antigen complex isformulated with a pharmaceutically acceptable excipient, such asphosphate buffered saline and injected subcutaneously into both flanksof BALB/c mice, which can be purchased from Jackson Laboratory, BarHarbor, Me. The amount of the particulate/recombinant HIV-160 gpglycoprotein complex is 10 μg. In a separate experiment, a pair of ageand sex matched BALB/c mice are injected with soluble recombinant HIV-1gp 160 glycoprotein (10 μg).

Approximately seven days following the injections, the mice aresacrificed and their spleen cells removed. To test for CTL activity,spleen cells (5×10⁶) are restimulated in vitro for five days withmitomycin C treated (50 μg/ml⁻¹, 45 min., 37° C.) BALB/c3T3 fibroblaststhat are transfected to express the whole gp160IIIB envelope protein, incomplete T cell medium.

After six days of culture, the cells are tested for their ability tolyse ⁵¹ Cr-labeled BALB/c3T3 transfectants expressing the wholegp160IIIB envelope protein or nongp160IIIB expressing BALB/c3T3 cellspulsed with 1 μm peptide 18IIB, which is a 15 residue synthetic peptidecorresponding to an immunodominant CTL epitope of HIV-1 gp 160.Takahashi, Proc. Natl. Acad. Sci., 85:3105-3109 (1988).

CTL's from mice immunized with the gp 160 glycoprotein-POLYBEAD complexare obtained that specifically lyse fibroblast targets either expressinggp160IIIB envelope protein or pulsed with the 15 residue 18IIIB peptide.In contrast, similarly restimulated spleen cells from mice immunizedwith soluble protein alone should fail to efficiently elicit a CTLresponse to either of these targets. This experiment suggests that itmay be possible to elicit human CTL by using particulate antigencomplexes of HIV antigens.

EXAMPLE 6

This Example demonstrates, using an accepted animal model of tumorimmunity, that the particulate protein antigen complexes of theinvention can be used to induce tumor immunity.

Experiment A

OVA-iron oxide particles and soluble OVA were prepared as described inExample 2A, formulated with phosphate buffered saline (PBS), andseparately injected subcutaneously into both flanks of C57Bl/6 mice(five per group), purchased from the Jackson Laboratory, Bar Harbor,Me., or bred at the Dana-Farber Cancer Institute (Boston, Mass.). Theamount of protein antigen complex injected was 75 μg. A second group ofmice was immunized with the same amount of soluble ovalbumin in saline,while a third group was not immunized. Ten days following theimmunizations, the mice were challenged subcutaneously with 1×10⁵ MO4cells, which are syngeneic B16 melanoma cells that have been transfectedwith cDNA encoding chicken ovalbumin and tumor growth was monitoredevery two days commencing on day six after challenge.

The resulting data are illustrated in FIG. 9. As illustrated in thisfigure, the mice that had been immunized with the OVA-iron oxide beads(open circles) showed virtually no tumor volume, while those that hadnot been immunized (open triangles), and those that had been immunizedwith the soluble OVA (closed circles) began to show significant increasein tumor volume on about day 12, with continued increase in tumor volumeover the next several days.

Experiment B

OVA-iron oxide particles and soluble OVA were again prepared asdescribed in Example 2A, formulated with phosphate buffered saline(PBS), and separately injected subcutaneously into both flanks ofC57Bl/6 mice (ten per group), purchased from the Jackson Laboratory, BarHarbor, Me, or bred at the Dana-Farber Cancer Institute (Boston, Mass.).The amount of protein antigen complex injected was 50 μg in saline. Asecond group of mice was not immunized. Seven days following theimmunizations, the mice were challenged subcutaneously with 1×10⁷ EG7cells, which are syngeneic EL4 lymphoma cells that have been transfectedwith cDNA encoding chicken ovalbumin. Survival of the challenged micewas monitored over a period of 140 days commencing immediately afterchallenge.

The data from this Experiment are set forth in FIG. 10. As illustratedin this figure, the mice that had been immunized with the OVA-iron oxidebeads (open triangles) showed 100% survival at 140 days post-challenge,while those that had not been immunized showed only about 35% survivalon day 140.

EXAMPLE 7

This Example demonstrates that a particulate-influenza complex inaccordance with the invention can confer protective immunity in a mammalfrom subsequent challenge with live influenza virus.

In this Example, a commercially available, formaldehyde-inactivatedinfluenza virus vaccine was covalently coupled to BIOMAG (TM) magnetizediron oxide particles, which are coated to provide amino groups. Thecomplexes were prepared substantially as described in Example 2A, exceptthat the antigen used was the Influenza Virus Vaccine, Trivalent, Type A(Wyeth Laboratories, Inc., Marietta, Pa.) instead of chicken ovalbumin.The complexes were formulated with phosphate buffered saline (PBS), andseparately injected subcutaneously into both flanks of Balb/C mice (fiveper group), purchased from the Jackson Laboratory, Bar Harbor, Me., orbred at the Dana-Farber Cancer Institute (Boston, Mass.). The amount ofprotein antigen complex injected was 50 μg in saline. A second group ofmice was not immunized. Seven days following the immunizations, the micewere challenged intranasally with live influenza virus (strain A/MEL/35,dose=4X LD50). Survival of the challenged mice was monitored over aperiod of 12 days commencing immediately after challenge.

The data from this Experiment are illustrated in FIG. 11. As illustratedin this figure, the mice that had been immunized with the inactivatedinfluenza-iron oxide beads (open circles) showed 100% survival at 12days post-challenge, while those that had not been immunized had alldied as of day 12.

EXAMPLE 8

This Example indicates that CD4+ T cells are not necessary foreffectiveness of the immunogens of the invention, and also that thecomplexes of the invention can be used to prime a CTL response inimmunodeficient animals.

In this experiment, C57Bl/6 or class II MHC-deficient mutant mice wereimmunized subcutaneously in both flanks with ovalbumin conjugated toiron oxide particles (25 μg) prepared as described in Example 2A. Theclass II mutant mice are animals which are lacking in class II moleculesdue to the disruption of IA^(b) through homologous recombination. See,Gusby, M. J., "Development of CD4+ T Cells In MHC Class II-DeficientMice'" Science, 253:1417-1420 (1991). These mice express no class IImolecules and hence fail to develop CD4+ T lymphocytes. Id. The mice canbe generated as described in Grusby et al, and are also availablecommercially from GenPharm International, (Mountain View, Calif.).

Seven days after immunization, one group of animals was reimmunized withthe same immunogen. Fourteen days after the initial immunization, allanimals were sacrificed, spleen cells removed, and restimulated in vitrowith irradiated with EG7 cells. More specifically, spleen cells (30×10⁶)were restimulated in vitro for five days with X-irradiated (20,000 rads)EG7-OVA cells (15×10⁶ cells), in 10 ml of media. EG7 is a EL4 tumor cellline transfected with OVA cDNA) (Moore et al, Cell, 54:777 (1988)).

After five days of culture, the cells were tested for their ability tolyse ⁵¹ Cr-labeled EL4 cells (BG) (C57BL/6, H2^(b) thymoma) or EG7cells, in accordance with conventional techniques.

The results of the experiment are illustrated in FIGS. 12A and 12B. Asshown in these figures, the particulate OVA antigen complexes primedCTL's in both the normal C57Bl/6 mice (MHC+/+) and the immunodeficientmutant mice (MHC -/-). Immunodeficient mice that had been immunizedtwice (FIG. 12B, Primed×2) showed greater response than did theimmunodeficient mice that had been primed only once (FIG. 12A,Primed×1).

These data indicate that use of the particulate protein antigencomplexes of the invention in vivo is a strong form of immunization.

I claim:
 1. A method for inducing a class I-restricted CTL response to a protein antigen in a mammal, comprising administering to a mammal a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a particulate-protein complexwherein the particulate protein complex comprises a particulate component having an average diameter ranging in size from about 0.5 μm to about 6 μm and having an outer surface linked to a non-replicating protein antigen derived from a pathogenic organism, with the proviso that the particulate component is not a prokaryotic or eukaryotic cell, a micellar, multimicellar, or liposome vesicle, or composed of detergents or lipids; wherein the protein antigen is taken up, processed and presented in association with MHC class I molecules by an antigen presenting cell; and wherein the particulate-protein complex is administered in an amount effective to induce a class I-restricted CTL response to the protein antigen in the mammal.
 2. A method according to claim 1, wherein the non-replicating protein antigen is linked to the particle component through a covalent linkage.
 3. A method according to claim 1, wherein the non-replicating protein antigen is linked to the particle component through a non-covalent linkage.
 4. A method according to claim 1, wherein the non-replicating protein antigen is a viral protein.
 5. A method according to claim 4, wherein the viral protein is derived from a virus selected from the group consisting of influenza viruses, retroviruses, POX viruses, Herpes viruses, respiratory syncytial viruses, rabies viruses, measles viruses, polio viruses and rotaviruses.
 6. A method according to claim 1, wherein the non-replicating protein antigen is a bacterial protein.
 7. A method according to claim 1, wherein the particulate component is an iron-oxide particle.
 8. A method according to claim 1, wherein the particulate component is a silica bead.
 9. A method according to claim 1, wherein the particulate component is a latex bead.
 10. A method according to claim 1, wherein the particulate component is a biocompatible polymer or copolymer selected from the group consisting of polysaccharides, proteins and oligosaccharides formed of two or more monosaccharides linked by glycosidic bonds.
 11. A method for inducing a class I-restricted CTL response to a protein antigen in a mammal, comprising administering to a mammal a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a particulate-protein complexwherein the particulate protein complex comprises a particulate component having an average diameter ranging in size from about 0.5 μm to about 6 μm and having an outer surface linked to a tumor antigen, with the proviso that the particulate component is not a prokaryotic or eukaryotic cell, a micellar, multimicellar, or liposome vesicle, or composed of detergents or lipids; wherein the antigen is taken up, processed and presented in association with MHC class I molecules by an antigen presenting cell; and wherein the particulate-protein complex is administered in an amount effective to induce a class I-restricted CTL response to the antigen in the mammal.
 12. A method according to claim 11, wherein the non-replicating protein antigen is linked to the particle component through a covalent linkage.
 13. A method according to claim 11, wherein the non-replicating protein antigen is linked to the particle component through a non-covalent linkage.
 14. A method according to claim 11, wherein the particulate component is an iron-oxide particle.
 15. A method according to claim 11, wherein the particulate component is a silica bead.
 16. A method according to claim 11, wherein the particulate component is a latex bead.
 17. A method according to claim 11, wherein the particulate component is a biocompatible polymer or copolymer selected from the group consisting of polysaccharides, proteins and oligosaccharides formed of two or more monosaccharides linked by glycosidic bonds.
 18. A method for inducing a class I-restricted CTL response to a protein antigen in a mammal, comprising administering to a mammal a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a particulate-protein complexwherein the particulate protein complex comprises a particulate component having an average diameter of about 10 μm and having an outer surface linked to a non-replicating protein antigen derived from a pathogenic organism, with the proviso that the particulate component is not a prokaryotic or eukaryotic cell, a micellar, multimicellar, or liposome vesicle, or composed of detergents or lipids; wherein the protein antigen is taken up, processed and presented in association with MHC class I molecules by an antigen presenting cell; and wherein the particulate-protein complex is administered in an amount effective to induce a class I-restricted CTL response to the protein antigen in the mammal.
 19. A method for inducing a class I-restricted CTL response to a bacterial protein antigen in a mammal, comprising administering to a mammal a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a particulate-protein complexwherein the particulate protein complex comprises a particulate component having an average diameter ranging in size from about 0.5 μm to about 6 μm and having an outer surface linked to a bacterial protein antigen, with the proviso that the particulate component is not a prokaryotic or eukaryotic cell, a micellar, multimicellar, or liposome vesicle, or composed of detergents or lipids; wherein the protein antigen is taken up, processed and presented in association with MHC class I molecules by an antigen presenting cell; and wherein the particulate-protein complex is administered in an amount effective to induce a class I-restricted CTL response to the protein antigen in the mammal.
 20. A method for inducing a class I-restricted CTL response to a viral protein antigen in a mammal, comprising administering to a mammal a pharmaceutical composition comprising a pharmaceutically acceptable excipient and particulate-protein complexwherein the particulate protein complex comprises a particulate component having an average diameter ranging in size from about 0.5 μm to about 6 μm and having an outer surface linked to a viral protein antigen, with the proviso that the particulate component is not a prokaryotic or eukaryotic cell, a micellar, multimicellar, or liposome vesicle, or composed of detergents or lipids; wherein the protein antigen is taken up, processed and presented in association with MHC class I molecules by an antigen presenting cell; and wherein the particulate-protein complex is administered in an amount effective to induce a class I-restricted CTL response to the protein antigen in the mammal. 